PROJECT SUMMARY/ABSTRACT. Mammalian cells possess immune defenses that act immediately to hinder the progression of pathogenic viral infection. In recent years, the Cristea laboratory and others have discovered that human cells can distinguish viral DNA from human DNA in the nuclei of infected cells. This finding was in contrast to the previous belief that sensing of viral DNA takes place only in subcellular compartments that lack human DNA (e.g., cytoplasm), and has provided an important new avenue of research for understanding cellular defense against nuclear-replicating DNA viruses. The principal discovery was the characterization of the interferon inducible protein IFI16 as a nuclear sensor of viral DNA and a crucial effector of intrinsic and innate immunity against nuclear-replicating herpesviruses, including herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). Our studies during infection with HSV-1 and HCMV have indicated that IFI16 binds viral double-stranded DNA in the nucleus through its HIN200 domains, oligomerizes through its pyrin domain, and activates the central cytoplasmic signaling axis, STING-TBK1-IRF3, to induce antiviral cytokines. However, how IFI16 initiates and propagates signals from the nucleus remains unknown. The goal of this proposal is to characterize the mechanism by which nuclear IFI16-mediated immune signals are propagated to this signaling axis upon herpesvirus infection. I will use a multidisciplinary approach that integrates proteomics with molecular virology, live cell microscopy, and biochemistry to define the mechanism of cellular immune response to herpesvirus infection. Performing these experiments with both HSV-1 and HCMV will help identify a broadly-relevant and conserved mechanism through which IFI16 signals. First, given my preliminary observation that IFI16 changes its subnuclear localization upon infection to associate with promyelocytic leukemia bodies (PML-NBs), I will investigate the means through which this localization is achieved. Specifically, I will conduct immunoaffinity isolations to assess the contributions of IFI16 structural motifs to mediating the interaction between IFI16 and PML-NBs. I will then use quantitative proteomic approaches to identify changes in the SUMOylation state of IFI16 during infection. Mutational analyses, immunofluorescence microscopy, and quantitative RT-PCR will be used next to assess the contribution of SUMOylation, SUMO interaction motifs (SIMs), and IFI16 structural properties to both the localization of IFI16 to PML-NBs and the downstream induction of antiviral cytokines. Lastly, I will characterize the function of IFI16 interactions that are unique to immune signaling during early infection. My preliminary studies showed that IFI16 associates with the antiviral IFIT 1/2/3 proteins. I will use live cell microscopy, knockdown assays, and antiviral cytokine measurements to assess the nuclear-cytoplasmic shuttling of IFIT proteins and their contribution to the propagation of the signals to STING. Altogether, this project will help elucidate the molecular mechanisms governing immune signal transmission, which is critical for understanding cellular immunity and for developing novel antiviral therapies.